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| United States Patent |
5,352,602
|
|
Yamada
, et al.
|
October 4, 1994
|
Isoamylase and process for producing the same
Abstract
An isoamylase, useful in the field of starch saccharification, having a
molecular weight of about 105,000, an isoelectric point of 6.4, an optimum
pH of 3.0 to 5.0, an optimum temperature of about 50.degree. C. and
exhibiting temperature stability at 45.degree. C..times.10 minutes and pH
stability at pH 3.5 to 6.0, and a process for producing the same
comprising cultivating an isoamylase-producing strain belonging to the
genus Xanthomonas and recovering the enzyme produced.
| Inventors:
|
Yamada; Yuzo (Shizuoka, JP);
Sato; Toshihiro (Shizuoka, JP);
Ohya; Takaichi (Aichi, JP)
|
| Assignee:
|
Amano Pharmaceutical Co., Ltd. (Aichi, JP)
|
| Appl. No.:
|
024072 |
| Filed:
|
March 1, 1993 |
Foreign Application Priority Data
| Current U.S. Class: |
435/210; 435/98; 435/910 |
| Intern'l Class: |
C12N 009/44; C12P 019/16 |
| Field of Search: |
435/210,98,910
|
References Cited
U.S. Patent Documents
| 3560345 | Feb., 1971 | Yokobayashi et al. | 435/210.
|
| 3716455 | Feb., 1973 | Ueda et al. | 435/210.
|
| 3723253 | Mar., 1973 | Yokobayashi et al. | 435/210.
|
| 3729380 | Apr., 1973 | Sugimoto et al. | 435/210.
|
| 3766014 | Oct., 1973 | Masuda et al. | 435/210.
|
| 4902622 | Feb., 1990 | Nakai et al. | 435/210.
|
| 5147795 | Sep., 1992 | Ara et al. | 435/210.
|
| Foreign Patent Documents |
| 1377064 | Dec., 1974 | GB | 435/210.
|
Other References
Ueda et al "Applied Microbiol" May 1967 pp. 49-496 vol. 5 No 3.
"World J. Microbiol Biotechnol" (1992) 8, 2, 102-5 Odibo et al.
Nogei Kagaku Kaishi, vol. 23, pp. 115-120, pp. 120-123 (1949).
Bull. Agr. Chem. Soc., vol. 19, No. 3, pp. 163-166 (1955).
FEBS Lett., vol. 12, pp. 96-100 (1970).
Biochem. Biophys. Acta, vol. 212, pp. 458-469 (1970).
|
Primary Examiner: Lilling; Herbert J.
Attorney, Agent or Firm: Sughrue, Mion, Zinn, Macpeak & Seas
Claims
What is claimed is:
1. Substantially pure isoamylase produced by cultivating an
isoamylase-producing Xanthomonas maltophilia strain, said substantially
pure isoamylase having the following enzymological and chemical
properties:
1) Substrate specificity: actively acts on glycogen, also acts on
amylopectin, hardly acts on pullulan,
2) Optimum pH: 3.0 to 5.0,
3) pH Stability: pH 3.5 to 6.0,
4) Optimum Temp.: around 50.degree. C.,
5) Temperature Stability: stable at 45.degree. C. for 10 minutes,
6) Isoelectric point: 6.4, and
7) Molecular weight: about 105,000, as measured by gel-filtration on
SUPEROSE 12.
2. A process for producing an isoamylase having the following enzymological
and chemical properties:
1) Substrate specificity: actively acts on glycogen, also acts on
amylopectin, hardly acts on pullulan,
2) Optimum pH: 3.0 to 5.0,
3) pH Stability: pH 3.5 to 6.0,
4) Optimum Temp.: around 50.degree. C.,
5) Temperature Stability: stable at 45.degree. C. for 10 minutes,
6) Isoelectric point: 6.4, and
7) Molecular weight: about 105,000, as measured by gel-filtration on
SUPEROSE 12,
comprising cultivating an isoamylase-producing strain belonging to the
genus Xanthomonas and recovering the isoamylase produced.
3. The process of claim 2, wherein the isoamylase-producing strain
belonging to the genus Xanthomonas is Xanthomonas maltophilia.
4. The substantially pure isoamylase of claim 1, wherein the strain of
Xanthomonas maltophilia is Xanthomonas maltophilia S-517.
5. The process of claim 3, wherein the strain of Xanthomonas maltophilia is
Xanthomonas maltophilia S-517.
6. The substantially pure isoamylase of claim 4, wherein the strain of
Xanthomonas maltophilia S-517 is that having accession number FERM
BP-4205.
7. The process of claim 5, wherein the strain of Xanthomonas maltophilia
S-517 is that having accession number FERM BP-4205.
Description
FIELD OF THE INVENTION
This invention relates to a substantially pure isoamylase and a process for
producing the same. The isoamylase of the present invention specifically
cleaves the .alpha. 1,6 bonds in glycogen and starches such as amylopectin
but does not act on pullulan and can be utilized in the field of starch
saccharification, for example, glucose production, fructose production and
maltose production.
BACKGROUND OF THE INVENTION
Known microorganisms capable of producing isoamylases include yeast (see
NOGEI KAGAKU KAISHI Vol. 23, pp. 115-120, pp. 120-123 (1949)), Cytophaga
(see FEBS Lett., Vol. 12, pp. 96-100 (1970)), and Pseudomonas (see
Biochem. Biophys. Acta, Vol. 212, pp. 458-469 (1970)).
These conventional isoamylases have several disadvantages in industrial
use. Isoamylase of yeast origin has poor heat stability. Isoamylase of
Cytophaga origin has insufficient heat stability and acid resistance. On
the other hand, isoamylase of Pseudomonas origin, while such does not have
the problem of heat stability and acid resistance, involves disadvantages
in productivity, i.e., need of a long cultivation time and low activity
attained. Thus, conventional isoamylases are unsatisfactory for use on an
industrial scale.
SUMMARY OF THE INVENTION
An object of the present invention is to provide an isoamylase with heat
stability and acid resistance.
Another object of the present invention is to provide a process for
producing an isoamylase which is heat stable and acid resistant at high
productivity.
As a result of extensive research, the inventors have found that a new
microorganism strain belonging to the genus Xanthomonas is capable of
producing an isoamylase which satisfies the object of the present
invention in large quantities. Enzymological examination revealed that the
recovered isoamylase is a novel isoamylase. The present invention has been
completed based on these findings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph showing the relationship between The optimum pH and
relative activity (%) of the enzyme according to the present invention.
.smallcircle. indicates a system using a sodium acetate-HCl buffer
solution; .DELTA. an acetate buffer solution; and .quadrature. a phosphate
buffer solution.
FIG. 2 is a graph showing the relationship between the pH and relative
activity (residual activity) (%), i.e., pH stability of the enzyme
according to the present invention. The symbols used have the same
meanings as in FIG. 1.
FIG. 3 is a graph showing the relationship between the optimum temperature
and relative activity (%) of the enzyme according to the present
invention.
FIG. 4 is a graph showing the relationship between the temperature and
relative activity (residual activity) (%), i.e., temperature stability, of
the enzyme according to the present invention.
DETAILED DESCRIPTION OF THE INVENTION
The newly discovered strain which can be used in the present invention was
isolated from nature. The microbiological characteristics of the strain
are as follows.
A. Morphology
1) Shape: straight rod
2) Size: 0.6 to 0.8.times.1.6 to 5.0 .mu.m
3) Motility: motile by polar multitrichous flagella. a few motile cells.
motile only at a low temperature and in the initial stage of culturing.
4) Spores: negative
B. Growth Characteristics on the Following Media
1) Meat extract-agar plate culture: small, circular, glossy, and pale
yellow colonies (diameter: less than 1 mm); slightly viscous
2) Litmus milk: peptonization (25 days)
C. Physiological Characteristics:
1) Nitrate reduction: positive
2) MR reaction: negative
3) VP reaction: negative
4) Indole production: negative
5) Hydrogen sulfide production: negative
6) Starch hydrolysis: negative
7) Utilization of citric acid: negative
8) Urease: negative
9) Oxidase: negative
10) Catalase: positive
11) Growable Range: Temp.: 7.degree. to 35 .degree. C. (optimum: 25.degree.
to 33 .degree. C.) pH: 4.5 to 7.7 (optimum: 5.0 to 5.7)
12) Behavior to oxygen: strictly aerobic
13) O-F test: oxidation
14) Gelatin liquefaction: positive
15) Production of pigment (King A medium): negative
16) Casein decomposition: negative
17) DNA decomposition: positive
18) Phenylalanine decomposition: negative
19) Production of acid from sugars:
1) Fructose: positive
2) Inositol: positive
3) Melibiose: positive
4) Maltose: positive
5) Cellobiose: positive
6) Lactose: positive
7) Xylose: positive
8) L-Arabinose: positive
9) Sucrose: positive
10) Trehalose: positive
11) Ribose: positive
12) Salicin: negative
13) Mannitol: negative
14) Sorbitol: negative
D. Other Characteristics:
1)Tolerance to sodium chloride: 0 to 3%
2) Decomposition of Tween 80: negative
3) Gluconic acid decomposition: negative
4) Esculin decomposition: positive
5) Acylamidase: negative
6) Arginine dihydrolase: negative
7) Lysine decarboxylase: negative
8) Ornithine decarboxylase: negative
9) Utilization of malonic acid: negative
10) Tyrosine decomposition: positive
11) ONPG test: negative
12) Poly-.beta.-hydroxybutyric acid accumulation: negative
13) Acid production from 10% Lactose: negative
14) Mucoid production: negative
15) Egg yolk reaction: negative
16) Growth in MacConkey agar medium: positive (weak)
17) Growth in SS (Salmonella-Shigella) medium: negative
18) Acid production in Sellers agar medium: negative
19) Acid production in TSI medium: negative
20) Ubiquinone: Q-8
The above microbiological properties were evaluated with reference to
Bergey's Manual of Systematic Bacteriology, Vol. 1 (1984). The strain was
considered to be a member of the family Pseudomonadaceae from the facts
that motility is present in the initial stage of low-temperature culture
and in a very small part of cells and that the cells have polar flagella.
However, from the fact that the strain does not grow at 37 .degree. C.,
has low tolerance to sodium chloride, prefers a slightly acidic region,
has no lysine decarboxylase activity, no oxidase activity or no
acylamidase activity, and does not accumulate poly-.beta.-hydroxybutyric
acid, it was finally identified to be Xanthomonas maltophilia. The strain
was designated Xanthomonas maltophilia S-517 and a culture was deposited
with Fermentation Research Institute, Agency of Industrial Science &
Technology, MITI, 1-3, Higaski, 1-chome Tsukuba-shi, Ibaraki-Ken, 305,
Japan receiving accession number FERM P-12779 (FERM BP-4205 under the
Budapest Treaty). The original deposit was made Feb. 19, 1992 and it was
transferred to a deposit under the Budapest Treaty on Feb. 24, 1993.
Carbon sources which can be present in the culture medium for production
and accumulation of an isoamylase produced by this microorganism include
sugars having an .alpha.-1,4 bond or an .alpha.-1,6 bond, such as starch,
soluble starch, dextrin, acid hydrolysis products of starch, enzyme
hydrolysis products of starch, maltose, isomaltose, and maltotriose.
Nitrogen sources which can be present in the medium include inorganic
nitrogen compounds, e.g., ammonium salts and nitrates, and organic
nitrogen compounds, e.g., urea, glutamic acid, aspartic acid, polypeptone,
corn steep liquor, meat extract, yeast extract, and hydrolysis products of
proteins. Natural media containing carbon sources and nitrogen sources at
moderate mixing ratios which mainly comprise rice flour or sweet potato
flour may also be used. In addition to the carbon sources and nitrogen
sources, the medium may contain appropriate inorganic salts as desired.
Cultivation is carried out by inoculating medium adjusted to pH 6 to 8 with
the above-described microorganism strain followed by shake culture or
aeration-agitation culture at a temperature of from 20.degree. to
35.degree. C. from 1 to 3 days. After cultivation, the microbial cells are
removed from the culture medium to obtain a supernatant liquor as a crude
enzyme liquid.
The crude enzyme liquid is then subjected to purification treatments, such
as ammonium sulfate fractionation and column chromatography on
DEAE-Sepharose CL-6B (produced by Pharmacia), CM-Sepharose CL-6B (produced
by Pharmacia), Superose 12 (produced by Pharmacia), etc., to obtain a high
purity isoamylase preparation showing a single band in SDS-polyacrylamide
gel electrophoresis (hereinafter abbreviated as SDS-PAGE). The
enzymological and chemical properties of the purified isoamylase are shown
below.
1) Substrate specificity: actively acts on glycogen, also acts on
amylopectin, hardly acts on pullulan as shown in Table 1 below.
TABLE 1
______________________________________
Initial
Substrate V (.mu.mol/min/U)
Velocity (%)
______________________________________
Glycogen 6.28 .times. 10.sup.-1
100
Amylopectin 2.18 .times. 10.sup.-1
34.7
Pullulan 8.45 .times. 10.sup.-3
1.3
______________________________________
2) Optimum pH: 3.0 to 5.0 (shown in FIG. 1)
3) pH Stability: pH 3.5 to 6.0 (40.degree. C..times.30 min, shown in FIG.
2)
4) Optimum Temp.: around 50.degree. C. (shown in FIG. 3)
5) Temperature Stability: stable when treated at 45.degree. C. for 10
minutes (shown in FIG. 4)
6) Isoelectric point (pI): 6.4 (Electrofocusing on Ampholine sucrose
density gradient)
7) Molecular weight: about 105,000 (measured by gel-filtration on Superose
12; two subunits of 58,000 and 50,000 appear in SDS-PAGE)
8) Influence of inhibitors: Influence of inhibitors and metal salts are
shown in Table 2 below.
TABLE 2
______________________________________
Percent
Concentration
Inhibition
Inhibitor (mM) (%)
______________________________________
N-Bromosuccinimide
0.01 100.0
Potassium iodoiodide
1 4.5
Acetyl monoiodide
5 34.6
Acetyl monoiodide
0.1 10.1
2,4-Dinitrofluorobenzene
1 2.1
Succinic anhydride
5 8.4
Glucono-1,5-lactone
10 8.0
2-Hydroxy-5-nitrobenzyl
0.1 14.3
bromide
EDTA 5 0
.beta.-Mercaptoethanol
10 31.7
PCMB 1 0
HgCl.sub.2 1 42.2
CuCl.sub.2 1 20.7
AgNO.sub.3 1 98.0
NaF 1 0
MgCl.sub.2 1 26.3
Ammonium molybdate
1 0
NaCl 1 0
______________________________________
Enzymological and chemical differences between the novel isoamylase
according to the present invention and known isoamylase preparations are
tabulated below. Reference is made to the literature cited above. A
"hyphen" in Table 3 below indicates that no data was given in the
literature.
TABLE 3
__________________________________________________________________________
Temp. Molecular
Inhibition by
Origin of
Optimum
pH Optimum
Stability
Weight
Metal Salt
Isoamylase
pH Stability
(.degree.C.)
(.degree.C.)
pI (.times. 10.sup.4)
(%)
__________________________________________________________________________
Yeast 6.2 -- 20 -- -- --
Cytophaga
5.5 -- 40 37 5.0-5.5
12 --
Pseudomonas
3-4 3-6 52 45 4.4 9.4-9.5
Mg.sup.+2
(0);
NaF (19);
PCMB
(34)
Xanthomonas
3-5 3.5-6.0
50 45 6.4 10.5 Mg.sup.+2
(26.3);
maltophilia NaF (0);
S-517 PCMB
(0)
(Invention)
__________________________________________________________________________
In contrast to the enzymological and chemical properties of known
isoamylase preparations, it is apparent from the data in Table 3 that the
enzyme of the present invention is a novel enzyme. That is, the enzyme of
the present invention has different isoelectric point and molecular weight
as compared to the enzyme derived from Pseudomonas, and the enzyme of the
present invention is not inhibited by NaF and PCMB but inhibited by Mg
ions, whereas the enzyme derived from Pseudomonas is not inhibited by Mg
ions but inhibited by NaF and PCMB.
Measurements of activity of the enzyme of the present invention were made
as follows in accordance with the method of Maruo-Kobayashi (see NIHON
NOGEI KAISHI, Vol. 23, pp. 115-120 (1949).
A reaction system consisting of 2.0 ml of a 1.0% soluble starch (glutinous
rice) solution, 0.4 ml of a 0.5M acetate buffer solution (pH=3.5), and 0.4
ml of an enzyme liquid was allowed to react at 40.degree. C. for 30
minutes. To the reaction system was added 0.4 ml of a 0.01N iodine
solution. After dilution with water to 10 ml, the absorbance at 610 nm was
measured. The enzyme activity on an absorbance increase of 0.1 within 1
hour was taken as 10 units.
The enzyme of the present invention can be utilized in the field of
saccharification by employing the conventional methods (cf. U.S. Pat. No.
3,795,584, GB-A-2,099,823, etc.).
The present invention is now illustrated in greater detail by reference to
the following Examples, but it should be understood that the present
invention is not deemed to be limited thereto. Unless otherwise indicated
herein, all parts, percents, ratios, and the like are by weight.
EXAMPLE 1
Medium (100 ml) containing 1.5% soluble starch, 0.5% meat extract, 0.3%
(NH.sub.4).sub.2 HPO.sub.4, 0.1% K.sub.2 HPO.sub.4, and 0.1% MgSO.sub.4.
7H.sub.2 O (starting pH: 5.5) was inoculated with Xanthomonas maltophilia
S-517 (FERM-P 12779), followed by spinner culture in a Sakaguchi flask at
30.degree. C. for 20 hours to produce an isoamylase in culture. The
microbial cells were removed by centrifugation, and the resulting
supernatant liquor (isoamylase activity: 305 unit/ml) was concentrated
using an ultrafilter. Cold ethyl alcohol was added to a concentration of
80% to precipitate the enzyme. The precipitate was collected by
centrifugation and lyophilized to obtain isoamylase powder. The recovery
of the crude enzyme from the supernatant liquor was 95%.
EXAMPLE 2
In a 30 l jar fermentor was placed 20 l of a medium containing 2.0%
maltose, 0.5% sodium glutamate, 0.5% yeast extract, 0.3% (NH.sub.4).sub.2
HPO.sub.4, 0.1% K.sub.2 HPO.sub.4,and 0.1% MgSO.sub.4. 7H.sub.2 O
(starting pH: 5.5) and the medium was inoculated with Xanthomonas
maltophilia S-517 (FERM-P 12779), followed by aeration culture at
30.degree. C. for 16 hours. The microbial cells were removed by
centrifugation, and the resulting supernatant liquor (isoamylase activity:
320 unit/ml) was 20-fold concentrated using an ultrafilter. The
concentrate was salted out with 60% saturated ammonium sulfate. The
precipitate was dissolved in a phosphate buffer solution (pH=6.0) and
passed through DEAE-Sepharose CL-6B. The effluent was then treated with
CM-Sepharose CL-6B and eluted with a 0.1M acetate buffer solution (pH=4.5)
with a concentration gradient. The combined active fraction was subjected
to gel filtration on Superose 12 to obtain 2.76.times.10.sup.5
unit/mg-protein of a purified enzyme preparation. The recovery of the
purified enzyme from the culture supernatant liquor was 83%.
According to the present invention, a novel isoamylase which is heat stable
and acid resistant can be produced in large quantities by culturing
Xanthomonas maltophilia in a short time. The present invention thus
provides an economical process for producing a useful isoamylase.
While the invention has been described in detail and with reference to
specific examples thereof, it will be apparent to one skilled in the art
that various changes and modifications can be made therein without
departing from the spirit and scope thereof.
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